Imprinting is an epigenetic phenomenon in which genes are uniquely "marked" in the parental gametes and differentially expressed post- fertilization, dependent upon the sex of the parent. Thus each imprinted genes has an active and inactive allele with transcription status dependent upon parent-of-origin. Several genetic diseases and cancers arise due to abnormal gene expression. The Angelman (AS) and Prader-Willi (PWS) syndromes are clinically distinct neurobehavioral disorders that represent the best example of this phenomenon in humans. Loss of paternally active 15qll-q13 genes results in PWS, whereas loss of a maternally active 15q11-q13 gene leads to AS. The molecular mechanisms by which individual alleles of imprinted genes are marked according to their parent- of-origin is not known, although distinct DNA methylation imprints have been identified for every imprinted gene that has been well characterized. It has been postulated that imprinting must first occur in the germ cells, beginning with erasure of the parental imprint and establishment of a new imprint depending upon the sex of the individual. Very little is known about how this process occurs in mammalian gametogenesis, particularly in humans. Therefore, the goals of this project are to determine the mechanisms and timing of imprinting in human gametogenesis specifically in the AS/PWS region of 15qll-q13. This will be accomplished by first separating the stages of male and female gametogenesis, and then: 1) assessing the expression pattern of imprinted genes in cells from various stages of gametogenesis; 2) identifying sites of differential methylation and examining their role in establishing imprinting; 3) identifying and characterizing sequence-specific DNA- protein interactions associated with the regulation of imprinted gene expression; and 4) assessing the state of histone acetylation across the 15q11-q13 region in meiosis in order to identify boundaries between maternally and paternally imprinted domains. These studies will allow us to directly address molecular mechanisms regarding the establishment and maintenance of imprinted gene expression.